1. Field of the Invention
The present invention relates generally to the fields of virology and gene therapy. More specifically, the present invention relates to the production of recombinant adenoviral vectors with modified fibers for the purpose of cell-specific targeting with the additional advantage of concomitant elimination of endogenous tropism.
2. Description of the Related Art
Recombinant adenovirus vectors are used in a number of gene therapy applications principally because of the high levels of gene transfer achievable with this approach both in vitro and in vivo. In addition, recombinant adenovirus vectors are distinguished from other available systems by their unique ability to accomplish in situ gene delivery to differentiated target cells in a variety of organ contexts.
Recombinant human adenovirus vectors of serotypes 2 (Ad2) and 5 (Ad5) have the ability to transfer genes to a range of cell types in vivo efficiently and have therefore been employed in a number of gene therapy approaches. However, it is not currently possible to exploit the full potential of adenovirus as a gene delivery vehicle exhibiting systemic stability following intravenous administration. Adenovirus-mediated delivery of a therapeutic gene selectively to target disease cells is precluded by the widespread distribution of primary cellular receptors for Ad2 and Ad5. In addition, it has recently been reported that a number of tissues which represent important targets for gene therapy, including the airway epithelium and primary tumors, express only low levels of primary adenovirus receptors and are thus poorly transduced by adenovirus vectors (1-4). Therefore, strategies are being developed to alter the tropism of the adenovirus vector to permit efficiently targeted gene delivery to specific cell types.
Two distinct, sequential steps are required for the entry of adenoviruses into susceptible cells. In the first step, Ad2 and Ad5 bind with high affinity to the primary cellular receptors, identified as the coxsackievirus and adenovirus receptor CAR (5-7), and the xcex12 domain of the major histocompatibility complex (MHC) class I protein (8). This binding occurs via the C-terminal knob domain of the adenovirus fiber protein (9,10). The second subsequent step is the internalization of the virion by receptor-mediated endocytosis potentiated by the interaction of Arg-Gly-Asp (RGD) peptide sequences in the penton base with secondary host cell receptors, integrins xcex1vxcex23 and xcex1vxcex25 (11,12). The virion then escapes from the endosome and localizes to the nuclear pore whereupon its genome is translocated to the nucleus.
Strategies to alter adenovirus tropism are based o n modifications of the viral capsid proteins to permit the recognition of alternative cell-specific receptors. Modification of adenovirus tropism by complexing adenovirus particles with bispecific conjugates that simultaneously ablate endogenous viral tropism and introduce novel tropism has been the main approach to date (4, 13-21). This approach to the generation of targeted adenovirus vectors suffers from a number of limitations that could be avoided by the direct genetic engineering of the viral capsid proteins to contain cell-targeting ligands. In this regard, the C-terminus of the adenovirus fiber protein can be modified to incorporate targeting motifs with specificity for cellular receptors (22-24).
In an alternative approach, targeting ligands can be incorporated within the so-called HI loop of the fiber knob (3, 25). These genetic modifications to the fiber protein have resulted in expanded tropism by redirecting adenovirus binding to alternative cellular receptors. However, these modified vectors also retain the ability to recognize the fiber receptor; native tropism has not been abolished.
Thus, to date, it has not, proven possible to employ genetic methods to engineer adenovirus vectors with specificity for a single target cell type. In addition to recognizing novel receptors, such vectors should also lack the ability to bind to the native primary adenovirus receptor. This can be accomplished either by site-directed mutagenesis of the fiber knob domain to eliminate the cell-binding site or by complete replacement of the fiber knob. However, an important consequence of the ablation of native adenovirus tropism is that it is not possible to propagate these vectors in standard, packaging cell lines that express the fiber receptor such as 293 (26) and 911 (27). Hence, it is necessary to construct alternative cell lines expressing novel primary receptors that can b e recognized by adenovirus vectors that fail to bind the fiber receptor.
The prior art is deficient in the lack of a fiber receptor-independent system for the propagation of adenoviral vectors and genetically modified adenovirus vectors with specificity for a single target cell type. The present invention fulfills this longstanding need and desire in the art.
Genetic modifications to the knob domain of the fiber protein of vectors based on human adenovirus serotype 5 (Ad5) have resulted in expanded tropism by redirecting adenovirus binding to alternative cellular receptors. However, native tropism has not been abolished to date: the vectors retain the ability to recognize the fiber receptor. An important consequence of strategies to ablate native adenovirus tropism is that it is not possible to propagate these modified vectors on standard cell lines that express the fiber receptor. Hence, it is necessary to construct alternative cell lines expressing novel primary receptors that can be recognized by adenovirus vectors that fail to bind the fiber receptor.
Towards this goal, two distinct artificial primary cellular receptors for Ad5 were generated. The extracellular domain of one of the synthetic receptors was derived from a single-chain antibody (sFv) with specificity for Ad5 knob, while the second receptor consisted of an icosapeptide identified from a phage display library by biopanning against Ad5 knob. Expression of either of these artificial receptors in fiber receptor-negative cells conferred susceptibility to Ad5 infection. Having shown the feasibility of engineering novel primary receptors for Ad5, an Ad5 vector was then genetically modified by incorporating six histidine (His) residues at the C-terminal of the fiber to serve as a motif specifically to mediate propagation of the vector. This vector could be amplified in fiber receptor-negative cells engineered to express an artificial surface receptor comprising an anti-His tag sFv. This novel fiber receptor-independent system for the propagation of adenovirus viruses is being used for the generation of vectors lacking native tropism.
In the present invention, there is provided a composition of matter comprising a method of propagating adenovirus independent of the ubiquitous adenovirus fiber protein receptor. An artificial, recombinant receptor is expressed in a host cell line which interacts with a surface protein on the virion other than the fiber protein gene. This enables the fiber protein receptor binding domain of the fiber protein to be altered to eliminate native tropism of the virus while still allowing the modified virus to be propagated in the host cell line.
In another embodiment of the current invention, the artificial receptor is specific for a recombinant protein marker expressed on the surface of the adenovirus.
In yet another embodiment of the current invention, the novel protein marker on the adenovirus surface is a recombinant protein tag added to the C-terminal end of the adenovirus fiber protein. This is recognized an artificial receptor containing an sFv specific for the said novel protein tag. In a preferred embodiment, the novel protein tag contains six C-terminal histidine and is recognized by an sFv from an anti-His tag monoclonal antibody.
The current invention includes the recombinant, artificial receptor designed to allow adenovirus propagation. This receptor comprises a signal peptide to direct the receptor to the cellular secretory pathway; a transmembrane domain for anchoring the receptor in the plasma membrane; and a protein binding domain specific for the protein marker with which the receptor is to associate. In the representative examples given herein, the artificial receptor is constructed with a signal peptide from the Ig-K leader sequence, and a transmembrane domain from the Platelet Derived Growth Factor Receptor (PDGFR). It also includes HA and Myc epitopes for immunological detection of the receptor. In the preferred embodiment described herein, the artificial contains an sFv derived from an anti-His tag monoclonal antibody.
The current invention also includes a recombinant adenovirus expressing an novel protein marker which can b e recognized by a specific artificial receptor. The adenovirus may also contain other genes such as therapeutic genes, transgenes for genomic modification, and marker genes for adenovirus detection.
In another embodiment of the current invention, the protein markers are fused to the C-terminal end of the adenovirus fiber protein. In a further embodiment, the protein marker fused to the C-terminal end of the fiber protein gene contains six C-terminal histidine residues. In the preferred embodiment of the current invention, the protein marker is 6xc3x97His, consisting of the peptide sequence (RGSHHHHHH) (SEQ ID NO:9) fused to the end of the fiber protein gene by the linker peptide (PSASASASAP) (SEQ ID NO:8).
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.